Hepatitis C virus asialoglycoproteins

ABSTRACT

Two Hepatitis C Virus envelope proteins (E1 and E2) are expressed without sialylation. Recombinant expression of these proteins in lower eukaryotes, or in mammalian cells in which terminal glycosylation is blocked, results in recombinant proteins which are more similar to native HCV glycoproteins. When isolated by GNA lectin affinity, the E1 and E2 proteins aggregate into virus-like particles.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 07/758,880,filed Sep. 13, 1991, now abandoned which is a continuation-in-part ofU.S. Ser. No. 07/611,419, filed Nov. 8, 1990, now abandoned, thediscosures of which are incorporated herein by reference.

DESCRIPTION

1. Technical Field

This invention relates to the general fields of recombinant proteinexpression and virology. More particularly, the invention relates toglycoproteins useful for diagnosis, treatment, and prophylaxis ofHepatitis C virus (HCV) infection, and methods for producing suchglycoproteins.

2. Background of the Invention

Non-A, Non-B hepatitis (NANBH) is a transmissible disease (or family ofdiseases) that is believed to be virally induced, and is distinguishablefrom other forms of virus-associated liver disease, such as those causedby hepatitis A virus (HAV), hepatitis B virus (HBV), delta hepatitisvirus (HDV), cytomegalovirus (CMV) or Epstein-Barr virus (EBV).Epidemiologic evidence suggests that there may be three types of NANBH:the water-borne epidemic type; the blood or needle associated type; andthe sporadically occurring community acquired type. The number ofcausative agents is unknown. However, a new viral species, hepatitis Cvirus (HCV) has recently been identified as the primary (if not only)cause of blood-borne NANBH (BB-NANBH). See for example PCT WO89/046699and U.S. patent application Ser. No. 07/355,002, filed May 18, 1989 nowabandoned. Hepatitis C appears to be the major form oftransfusion-associated hepatitis in a number of countries or regions,including the United States, Europe, and Japan. There is also evidenceimplicating HCV in induction of hepatocellular carcinoma. Thus, a needexists for an effective method for preventing and treating HCVinfection.

The demand for sensitive, specific methods for screening and identifyingcarriers of HCV and HCV-contaminated blood or blood products issignificant. Post-trans-fusion hepatitis (PTH) occurs in approximately10% of transfused patients, and HCV accounts for up to 90% of thesecases. The major problem in this disease is the frequent progression tochronic liver damage (25-55%).

Patient care, as well as the prevention of transmission of HCV by bloodand blood products or by close personal contact, requires reliablediagnostic and prognostic tools to detect nucleic acids, antigens andantibodies related to HCV. In addition, there is also a need foreffective vaccines and immunotherapeutic therapeutic agents for theprevention and/or treatment of the disease.

HCV appears in the blood of infected individuals at very low ratesrelative to other infectious viruses, which makes the virus verydifficult to detect. The low viral burden is probably the primary reasonthat the causative agent of NANB hepatitis went so long undetected. Eventhough it has now been cloned, HCV still proves difficult to culture andpropagate. Accordingly, there is a strong need for recombinant means ofproducing diagnostic/therapeutic/prophylactic HCV proteins.

DISCLOSURE OF THE INVENTION

It has been found that two HCV proteins, E1 and E2, appear to bemembrane associated asialoglycoproteins when expressed in recombinantsystems. This is surprising because glycoproteins do not usually remainin mannose-terminated form in mammals, but are further modified withother carbohydrates: the mannose-terminated form is typically onlytransient. In the case of E1 and E2 (as expressed in our systems), theasialoglycoprotein appears to be the final form. E1 (envelope protein 1)is a glycoprotein having a molecular weight of about 35 kD which istranslated from the predicted E1 region of the HCV genome. E2 (envelopeprotein 2) is a glycoprotein having a molecular weight of about 72 kDwhich is translated from the predicted NSl (non-structural protein 1)region of the HCV genome, based on the flaviviral model of HCV. As viralglycoproteins are often highly immunogenic, E1 and E2 are primecandidates for use in immunoassays and therapeutic/prophylacticvaccines.

The discovery that E1 and E2 are not sialylated is significant. Theparticular form of a protein often dictates which cells may serve assuitable hosts for recombinant expression. Prokaryotes such as E. colido not glycosylate proteins, and are generally not suitable forproduction of glycoproteins for use as antigens because glycosylation isoften important for full antigenicity, solubility, and stability of theprotein. Lower eukaryotes such as yeast and fungi glycosylate proteins,but are generally unable to add terminal sialic acid residues to thecarbohydrate complexes. Thus, yeast-derived proteins may beantigenically distinct from their natural (non-recombinant)counterparts. Expression in mammalian cells is preferred forapplications in which the antigenicity of the product is important, asthe glycosylation of the recombinant protein should closely resemblethat of the wild viral proteins.

New evidence indicates that the HCV virus may gain entry to host cellsduring infection through either the asialoglycoprotein receptor found onhepatocytes, or through the mannose receptor found on hepaticendothelial cells and macrophages (particularly Kupffer cells).Surprisingly, it has been found that the bulk of natural E1 and E2 donot contain terminal sialic acid residues, but are onlycore-glycosylated. A small fraction additionally contains terminalN-acetylglucosamine. Accordingly, it is an object of the presentinvention to provide HCV envelope glycoproteins lacking all orsubstantially all terminal sialic acid residues.

Another aspect of the invention is a method for producing asialo-E1 orE2, under conditions inhibiting addition of terminal sialic acid, e.g.,by expression in yeast or by expression in mammalian cells usingantibiotics to facilitate secretion or release.

Another aspect of the invention is a method for purifying E1 or E2 byaffinity to lectins which bind terminal mannose residues or terminalN-acetylglucosamine residues.

Another aspect of the invention is an immunogenic composition comprisinga recombinant asialoglycoprotein selected from the group consisting ofHCV E1 and E2 in combination with a pharmaceutically acceptable vehicle.One may optionally include an immunological adjuvant, if desired.

Another aspect of the invention is an immunoassay reagent, comprising arecombinant asialoglycoprotein selected from the group consisting of HCVE1 and E2 in combination with a suitable support. Another immunoassayreagent of the invention comprises a recombinant asialoglycoproteinselected from the group consisting of HCV E1 and E2 in combination witha suitable detectable label.

Another aspect of the invention concerns dimers and higher-orderaggregates of E1 and/or E2. One species of the invention is an E2complex. Another species of the invention is an E1:E2 heterodimer.

Another aspect of the invention is an HCV vaccine composition comprisingE1:E2 aggregates and a pharmaceutically acceptable carrier.

Another aspect of the invention is a method for purifying E1:E2complexes.

MODES OF CARRYING OUT THE INVENTION A. DEFINITIONS

The term “asialoglycoprotein” refers to a glycosylated protein which issubstantially free of sialic acid moieties. Asialoglycoproteins may beprepared recombinantly, or by purification from cell culture or naturalsources. Presently preferred asialoglycoproteins are derived from HCV,preferably the glycoproteins E1 and E2, most preferably recombinant E1and E2 (rE1 and rE2). A protein is “substantially free” of sialic acidwithin the scope of this definition if the amount of sialic acidresidues does not substantially interfere with binding of theglycoprotein to mannose-binding proteins such as GNA. This degree ofsialylation will generally be obtained where less than about 40% of thetotal N-linked carbohydrate is sialic acid, more preferably less thanabout 30%, more preferably less than about 20%, more preferably lessthan about 10%, more preferably less than about 5%, and most preferablyless than about 2%.

The term “E1” as used herein refers to a protein or polypeptideexpressed within the first 400 amino acids of an HCV polyprotein,sometimes referred to as the E or S protein. In its natural form it is a35 kD glycoprotein which is found strongly membrane-associated. In mostnatural HCV strains, the E1 protein is encoded in the viral polyproteinfollowing the C (core) protein. The E1 protein extends fromapproximately amino acid 192 to about aa383 of the full-lengthpolyprotein. The term “E1” as used herein also includes analogs andtruncated mutants which are immunologically crossreactive with naturalE1.

The term “E2” as used herein refers to a protein or polypeptideexpressed within the first 900 amino acids of an HCV polyprotein,sometimes referred to as the NS1 protein. In its natural form it is a 72kD glycoprotein which is found strongly membrane-associated. In mostnatural HCV strains, the E2 protein follows the E1 protein. The E2protein extends from approximately aa384 to about aa820. The term “E2”as used herein also includes analogs and truncated mutants which areimmunologically crossreactive with natural E2.

The term “aggregate” as used herein refers to a complex of E1 and/or E 2containing more than one E1 or E2 monomer. E1:E1 dimers, E2: E2 dimers,and E1:E2 heterodimers are all “aggregates” within the scope of thisdefinition. Compositions of the invention may also include largeraggregates, and may have molecular weights in excess of 800 kD.

The term “particle” as used herein refers to an E1 , E2, or E1/E2aggregate visible by electron microscopy and having a dimension of atleast 20 nm. Preferred particles are those having a roughly sphericalappearance and a diameter of approximately 40 mn by electron microscopy.

The term “purified” as applied to proteins herein refers to acomposition wherein the desired protein comprises at least 35% of thetotal protein component in the composition. The desired proteinpreferably comprises at least 40%, more preferably at least about 50%,more preferably at least about 60%, still more preferably at least about70%, even more preferably at least about 80%, even more preferably atleast about 90%, and most preferably at least about 95% of the totalprotein component. The composition may contain other compounds such ascarbohydrates, salts, lipids, solvents, and the like, without affectingthe determination of percentage purity as used herein. An “isolated” HCVasialoglycoprotein intends an HCV asialoglycoprotein composition whichis at least 35% pure. “Mannose-binding protein” as used herein intends alectin or other protein which specifically binds to proteins havingmannose-terminated glycosylation (e.g., asialo-glycoproteins), forexample, mannose-binding lectins, antibodies specific formannose-terminated glycosylation, mannose receptor protein (R. A. B.Ezekowitz et al., J Exp Med (1990) 176:1785-94), asialoglycoproteinreceptor proteins (H. Kurata et al., J Biol Chem (1990) 265:11295-98),serum mannose-binding protein (I. Schuffenecker et al., Cytogenet CellGenet (1991) 56:99-102; K. Sastry et al., J Immunol (1991) 147:692-97),serum asialoglycoprotein-binding protein, and the like. Mannose-bindinglectins include, for example, GNA, Concanavalin A (ConA), and otherlectins with similar binding properties.

The term “GNA lectin” refers to Galanthus nivalus agglutinin, acommercially available lectin which binds to mannose-terminatedglycoproteins.

A “recombinant” glycoprotein as used herein is a glycoprotein expressedfrom a recombinant polynucleotide, in which the structural gene encodingthe glycoprotein is expressed under the control of regulatory sequencesnot naturally adjacent to the structural gene, or in which thestructural gene is modified. For example, one may form a vector in whichthe E1 structural gene is placed under control of a functional fragmentof the yeast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter.A presently preferred promoter for use in yeast is the hybrid ADH2/GAPpromoter described in U.S. Pat. No. 4,880,734 (incorporated herein byreference), which employs a fragment of the GAPDH promoter incombination with the upstream activation sequence derived from alcoholdehydrogenase 2. Modifications of the structural gene may includesubstitution of different codons with degenerate codons (e.g., toutilize host-preferred codons, eliminate or generate restriction enzymecleavage sites, to control hairpin formation, etc.), and substitution,insertion or deletion of a limited number of codons encoding differentamino acids (preferably no more than about 10%, more preferably lessthan about 5% by number of the natural amino acid sequence should bealtered), and the like. Similarly, a “recombinant” receptor refers to areceptor protein expressed from a recombinant polynucleotide, in whichthe structural gene encoding the receptor is expressed under the controlof regulatory sequences not naturally adjacent to the structural gene,or in which the structural gene is modified.

The term “isolated polypeptide” refers to a polypeptide which issubstantially free of other HCV viral components, particularlypolynucleotides. A polypeptide composition is “substantially free” ofanother component if the weight of the polypeptide in the composition isat least 70% of the weight of the polypeptide and other componentcombined, more preferably at least about 80%, still more preferablyabout 90%, and most preferably 95% or greater. For example, acomposition containing 100 μg/ml E1 and only 3 μg/ml other HCVcomponents (e.g., DNA, lipids, etc.) is substantially free of “other HCVviral components”, and thus is a composition of an isolated polypeptidewithin the scope of this definition.

The term “secretion leader” refers to a polypeptide which, when encodedat the N-terminus of a protein, causes the protein to be secreted intothe host cell's culture medium following translation. The secretionleader will generally be derived from the host cell employed. Forexample, suitable secretion leaders for use in yeast include theSaccharomyces cerevisiae α-factor leader (see U.S. Pat. No. 4,870,008,incorporated herein by reference).

The term “lower eukaryote” refers to host cells such as yeast, fungi,and the like. Lower eukaryotes are generally (but not necessarily)unicellular. Preferred lower eukaryotes are yeasts, particularly specieswithin Saccharomyces, Schizosaccharomyces, Kluveromyces, Pichia,Hansenula, and the like. Saccharomyces cerevisiae, S. carlsbergensis andK. lactis are the most commonly used yeast hosts, and are convenientfungal hosts.

The term “higher eukaryote” refers to host cells derived from higheranimals, such as mammals, reptiles, insects, and the like. Presentlypreferred higher eukaryote host cells are derived from Chinese hamster(e.g., CHO), monkey (e.g., COS cells), human, and insect (e.g.,Spodoptera frugiperda). The host cells may be provided in suspension orflask cultures, tissue cultures, organ cultures, and the like.

The term “calcium modulator” refers to a compound capable ofsequestering or binding calcium ions within the endoplasmic reticulum,or affects calcium ion concentration within the ER by its effect oncalcium regulatory proteins (e.g., calcium channel proteins, calciumpumps, etc.). Suitable calcium modulators include, for examplethapsigargin, EGTA (ethylene glycol bis[β-aminoethyl ether]N,N,N′,N′-tetraacetic acid). The presently preferred modulator isthapsigargin (see e.g., O. Thastrup et al, Proc Nat Acad Sci USA (1990)87:2466-70).

The term “immunogenic” refers to the ability of a substance to cause ahumoral and/or cellular immune response, whether alone or when linked toa carrier, in the presence or absence of an adjuvant. “Neutralization”refers to an immune response that blocks the infectivity, eitherpartially or fully, of an infectious agent. A “vaccine” is animmunogenic composition capable of eliciting protection against HCV,whether partial or complete, useful for treatment of an individual.

The term “biological liquid” refers to a fluid obtained from anorganism, such as serum, plasma, saliva, gastric secretions, mucus, andthe like. In general, a biological liquid will be screened for thepresence of HCV particles. Some biological fluids are used as a sourceof other products, such as clotting factors (e.g., Factor VII:C), serumalbumin, growth hormone, and the like. In such cases, it is importantthat the source biological fluid be free of contamination by virus suchas HCV.

B. GENERAL METHOD

The E1 region of the HCV genome is described in EP 388,232 as region“E”, while E2 is described as “NS 1.” The E1 region comprisesapproximately amino acids 192-383 in the full-length viral polyprotein.The E2 region comprises approximately amino acids 384-820. The completesequences of prototypes of these proteins (strain HCV-1) are availablein the art (see EP 388,232), as are general methods for cloning andexpressing the proteins. Both E1 and E2 may be expressed from apolynucleotide encoding the first 850-900 amino acids of the HCVpolyprotein (see, e.g., SEQ ID NO:3): post-translational processing inmost eukaryotic host cells cleaves the initial polyprotein into C, E1,and E2. One may truncate the 5′ end of the coding region to reduce theamount of C protein produced.

Expression of asialoglycoproteins may be achieved by a number ofmethods. For example, one may obtain expression in lower eukaryotes(such as yeast) which do not normally add sialic acid residues toglycosylated proteins. In yeast expression systems, it is presentlypreferred to employ a secretion leader such as the S. cerevisiaeα-factor leader, so that the protein is expressed into the culturemedium following translation. It is also presently preferred to employglycosylation-deficient mutants such as pmr1, as these mutants supplyonly core glycosylation, and often secrete heterologous proteins withhigher efficiency (H. K. Rudolph et al, Cell (1989) 58: 133-45).Alternatively, one may employ other species of yeast, such as Pichiapastotis, which express glycoproteins containing 8-9 mannose residues ina pattern believed to resemble the core glycosylation pattern observedin mammals and S. cerevisiae.

Alternatively, one may arrange expression in mammalian cells, and blockterminal glycosylation (addition of sialic acid). Recombinant constructswill preferably include a secretion signal to insure that the protein isdirected toward the endoplasmic reticulum. Transport to the golgiappears to be blocked by E1 and E2 themselves: highlevel expression ofE1 or E2 in mammalian cells appears to arrest secretion of all cellularproteins at the endoplasmic reticulum or cis golgi. One may additionallyemploy a glycosylation defective mutant. See for example, P. Stanley,Ann Rev Genet (1984) 18:525-52. In the event a glycosylation ortransport mutant expresses E1 or E2 with sialylation, the terminalsialic acid residues may be removed by treatment with neuraminidase.

Yield should be further increased by use of a calcium modulator toobtain release of protein from within the endoplasmic reticulum.Suitable modulators include thapsigargin, EGTA, and A23817 (see e.g., O.Thastrup et al, Proc Nat Acad Sci USA (1990) 87:2466-70). For example,one may express a large amount of E1 or E2 intracellularly in mammaliancells (e.g., CHO, COS, HeLa cells, and the like) by transfection with arecombinant vaccinia virus vector. After allowing time for proteinexpression and accumulation in the endoplasmic reticulum, the cells areexposed to a calcium modulator in concentration large enough to causerelease of the ER contents. The protein is then recovered from theculture medium, which is replaced for the next cycle.

Additionally, it may be advantageous to express a truncated form of theenvelope protein. Both E1 and E2 appear to have a highly hydrophobicdomain, which apparently anchors the protein within the endoplasmicreticulum and prevents efficient release. Thus, one may wish to deleteportions of the sequence found in one or more of the regions aal170-190,aa260-290 or aa330-380 of E1 (numbering from the beginning of thepolyprotein), and aa660-830 of E2 (see for example FIG. 20-1 of EP388,232). It is likely that at least one of these hydrophobic domainsforms a transmembrane region which is not essential for antigenicity ofthe protein, and which may thus be deleted without detrimental effect.The best region to delete may be determined by conducting a small numberof deletion experiments within the skill of the ordinary practitioner.Deletion of the hydrophobic 3′ end of E2 results in secretion of aportion of the E2 expressed, with sialylation of the secreted protein.

One may use any of a variety of vectors to obtain expression. Lowereukaryotes such as yeast are typically transformed with plasmids usingthe calcium phosphate precipitation method, or are transfected with arecombinant virus. The vectors may replicate within the host cellindependently, or may integrate into the host cell genome. Highereukaryotes may be transformed with plasmids, but are typically infectedwith a recombinant virus, for example a recombinant vaccinia virus.Vaccinia is particularly preferred, as infection with vaccinia haltsexpression of host cell proteins. Presently preferred host cells includeHeLa and plasmacytoma cell lines. In the present system, this means thatE1 and E2 accumulate as the major glycosylated species in the host ER.As the rE1 and rE2 will be the predominant glycoproteins which aremannose-terminated, they may easily be purified from the cells by usinglectins such as Galanthus nivalus agglutinin (GNA) which bind terminalmannose residues.

Proteins which are naturally expressed as mannose-terminatedglycoproteins are relatively rare in mammalian physiology. In mostcases, a mammalian glycoprotein is mannose-terminated only as atransient intermediate in the glycosylation pathway. The fact that HCVenvelope proteins, expressed recombinantly, contain mannose-tenninatedglycosylation or (to a lesser degree) N-acetylglucosamine means that HCVproteins and whole virions may be separated and partially purified fromendogenous proteins using lectins specific for terminal mannose orN-acetylglucosamine. The recombinant proteins appear authentic, and arebelieved essentially identical to the envelope proteins found in themature, free virion, or to a form of cell-associated envelope protein.Thus, one may employ lectins such as GNA for mannose-terminatedproteins, and WGA (wheat germ agglutinin) and its equivalents forN-acetylglucosamine-terminated proteins. One may employ lectins bound toa solid phase (e.g., a lectin-Sepharose® column) to separate E1 and E2from cell culture supernatants and other fluids, e.g., for purificationduring the production of antigens for vaccine or immunoassay use.

Alternatively, one may provide a suitable lectin to isolate E1, E2, orHCV virions from fluid or tissue samples from subjects suspected of HCVinfection. As man-nose-terminated glycoproteins are relatively rare,such a procedure should serve to purify the proteins present in asample, substantially reducing the background. Following binding tolectin, the HCV protein may be detected using anti-HCV antibodies. Ifwhole virions are present, one may alternatively detect HCV nucleicacids using PCR techniques or other nucleic acid amplification methodsdirected toward conserved regions of the HCV genome (for example, the 5′non-coding region). This method permits isolation and characterizationof differing strains of HCV without regard for antigenic drift orvariation, e.g., in cases where a new strain is not immunologicallycrossreactive with the strain used for preparing antibodies. There aremany other ways to take advantage of the unique recognition ofmannose-terminated glycoproteins by particular lectins. For example, onemay incubate samples suspected of containing HCV viriors or proteinswith biotin or avidin-labeled lectins, and precipitate theprotein-lectin complex using avidin or biotin. One may also use lectinaffinity for HCV proteins to target compounds to virions for therapeuticuse, for example by conjugating an antiviral compound to GNA.Alternatively, one may use suitable lectins to remove mannose-terminatedglycoproteins from serum or plasma fractions, thus reducing oreliminating the risk of HCV contamination.

It is presently preferred to isolate E1 and/or E2 asialoglycoproteinsfrom crude cell lysates by incubation with an immobilizedmannose-binding protein, particularly a lectin such as ConA or GNA.Cells are lysed, e.g., by mechanical disruption in a hypotonic bufferfollowed by centrifugation to prepare a post-nuclear lysate, and furthercentrifuged to obtain a crude microsomal membrane fraction. The crudemembrane fraction is subsequently solubilized in a buffer containing adetergent, such as Triton X-100, NP40, or the like. This detergentextract is further clarified of insoluble particulates bycentrifugation, and the resulting clarified lysate incubated in achromatography column comprising an immobilized mannose-binding protein,preferably GNA bound to a solid support such as agarose or Sepharose®for a period of time sufficient for binding, typically 16 to 20 hours.The suspension is then applied to the column until E1/E2 begins toappear in the eluent, then incubated in the column for a period of timesufficient for binding, typically about 12-24 hours. The bound materialis then washed with additional buffer containing detergent (e.g., TritonX-100, NP40, or the like), and eluted with mannose to provide purifiedasialoglycoprotein. On elution, it is preferred to elute only untilprotein begins to appear in the eluate, at which point elution is haltedand the column permitted to equilibrate for 2-3 hours before proceedingwith elution of the protein. This is believed to allow sufficient timefor the slow off-rate expected of large protein aggregates. In caseswherein E1 and E2 are expressed together in native form (i.e., withouttruncation of the membrane-binding domain), a substantial fraction ofthe asialoglycoproteins appear as E1:E2 aggregates. When examined byelectron microscopy, a significant portion of these aggregates appear asroughly spherical particles having a diameter of about 40 nm, which isthe size expected for intact virus. These particles appear to beself-assembling subviral particles. These aggregates are expected toexhibit a quaternary structure very similar to the structure ofauthentic HCV virion particles, and thus are expected to serve as highlyimmunogenic vaccines.

The E1/E2 complexes may be further purified by gel chromatography on abasic medium, for example, Fractogel-DEAE or DEAE-Sepharose®. UsingFractogel-DEAE gel chromatography, one may obtain E1/E2 complexes ofapproximately 60-80% purity. One may further purify E1 by treatment withlysine protease, because E1 has 0-1 Lys residues. Treatment of thecomplex with lysine protease destroys E2, and permits facile separationof E1.

The tissue specificity of HCV, in combination with the observation thatHCV envelope glycoproteins are mannose-terminated, suggests that thevirus employs the mannose receptor or the asialoglycoprotein receptor(ASGR) in order to gain entry into host cells. Mannose receptors arefound on macrophages and hepatic sinusoidal cells, while the ASGR isfound on parenchymal hepatocytes. Thus, it should be possible to cultureHCV by employing host cells which express one or both of thesereceptors. One may either employ primary cell cultures which naturallyexpress the receptor, using conditions under which the receptor ismaintained, or one may transfect another cell line such as HeLa, CHO,COS, and the like, with a vector providing for expression of thereceptor. Cloning of the mannose receptor and its transfection andexpression in fibroblasts has been demonstrated by M. E. Taylor et al, JBiol Chem (1990) 265:12156-62. Cloning and sequencing of the ASGR wasdescribed by K. Drickamer et al, J Biol Chem (1984) 259:770-78 and M.Spiess et al, Proc Nat Acad Sci USA (1985) 82:6465-69; transfection andexpression of functional ASGR in rat HTC cells was described by M.McPhaul and P. Berg, Proc Nat Acad Sci USA (1986) 83:8863-67 and M.McPhaul and P. Berg, Mol Cell Biol (1987) 7:1841-47. Thus, it ispossible to transfect one or both receptors into suitable cell lines,such as CHO, COS, HeLa, and the like, and to use the resulting cells ashosts for propagation of HCV in culture. Serial passaging of HCV in suchcultures should result in development of attenuated strains suitable foruse as live vaccines. It is presently preferred to employ animmortalized cell line transfected with one or both recombinantreceptors.

Immunogenic compositions can be prepared according to methods known inthe art. The present compositions comprise an immunogenic amount of apolypeptide, e.g., E1, E2, or E1/E2 particle compositions, usuallycombined with a pharmaceutically acceptable carrier, preferably furthercomprising an adjuvant. If a “cocktail” is desired, a combination of HCVpolypeptides, such as, for example, E1 plus E2 antigens, can be mixedtogether for heightened efficacy. The virus-like particles of E1/E2aggregates are expected to provide a particularly useful vaccineantigen. Immunogenic compositions may be administered to animals toinduce production of antibodies, either to provide a source ofantibodies or to induce protective immunity in the animal.

Pharmaceutically acceptable carriers include any carrier that does notitself induce the production of antibodies harmful to the individualreceiving the composition. Suitable carriers are typically large, slowlymetabolized macromolecules such as proteins, polysaccharides, polylacticacids, polyglycolic acids, polymeric amino acids, amino acid copolymers;and inactive virus particles. Such carriers are well known to those ofordinary skill in the art.

Preferred adjuvants to enhance effectiveness of the composition include,but are not limited to: aluminum hydroxide (alum),N-acetyl-muramyl-L-threonyl-D-isogluta-mine (thr-MDP) as found in U.SPat. No. 4,606,918, N-acetyl-normuramyl-L-alanyl-D-isoglutamine(nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(MTP-PE) and RIBI, which contains three components extracted frombacteria, monophosphoryl lipid A, trehalose dimycolate, and cell wallskeleton (MPL+TDM+CWS) in a 2% squalene/Tween® 80 emulsion.Additionally, adjuvants such as Stimulon (Cambridge Bioscience,Worcester, Mass.) may be used. Further, Complete Freund's Adjuvant (CFA)and Incomplete Freund's Adjuvant (IFA) may be used for non-humanapplications and research purposes.

The immunogenic compositions typically will contain pharmaceuticallyacceptable vehicles, such as water, saline, glycerol, ethanol, etc.Additionally, auxiliary substances, such as wetting or emulsifyingagents, pH buffering substances, and the like, may be included in suchvehicles.

Typically, the immunogenic compositions are prepared as injectables,either as liquid solutions or suspensions; solid forms suitable forsolution in, or suspension in, liquid vehicles prior to injection mayalso be prepared. The preparation also may be emulsified or encapsulatedin liposomes for enhanced adjuvant effect.

Immunogenic compositions used as vaccines comprise an immunologicallyeffective amount of the HCV polypeptide, as well as any other of theabove-mentioned components, as needed. “Immunologically effectiveamount”, means that the administration of that amount to an individual,either in a single dose or as part of a series, is effective fortreatment, as defined above. This amount varies depending upon thehealth and physical condition of the individual to be treated, thetaxonomic group of individual to be treated (e.g., nonhuman primate,primate, etc.), the capacity of the individual's immune system tosynthesize antibodies, the degree of protection desired, the formulationof the vaccine, the treating doctor's assessment of the medicalsituation, the strain of infecting HCV, and other relevant factors. Itis expected that the amount will fall in a relatively broad range thatcan be determined through routine trials.

The self-assembling E1/E2 aggregates may also serve as vaccine carriersto present heterologous (non-HCV) haptens, in the same manner asHepatitis B surface antigen (See European Patent Application 174,444).In this use, the E1/E2 aggregates provide an immunogenic carrier capableof stimulating an immune response to haptens or antigens conjugated tothe aggregate. The antigen may be conjugated either by conventionalchemical methods, or may be cloned into the gene encoding E1 and/or E2at a location corresponding to a hydrophilic region of the protein.

The immunogenic compositions are conventionally administeredparenterally, typically by injection, for example, subcutaneously orintramuscularly. Additional formulations suitable for other modes ofadministration include oral formulations and suppositories. Dosagetreatment may be a single dose schedule or a multiple dose schedule. Thevaccine may be administered in conjunction with other immunoregulatoryagents.

C. EXAMPLES

The examples presented below are provided as a further guide to thepractitioner of ordinary skill in the art, and are not to be construedas limiting the invention in any way.

Example 1 (Cloning and Expression)

(A) Vectors were constructed from plasmids containing the 5′ portion ofthe HCV genome, as described in EP 318,216 and EP 388,232. CassetteHCV(S/13) contains a StuI-BglII DNA fragment encoding the 5′ end of thepolyprotein from Met₁ up to Leu₉₀₆, beginning at nucleotide −63 relativeto Met₁. This includes the core protein (C), the E1 protein (alsosometimes referred to as S), the E2 protein (also referred to as NS1),and a 5′ portion of the NS2a region. Upon expression of the construct,the individual C, E1 and E2 proteins are produced by proteolyticprocessing.

Cassette HCV(A/B) contains a ApaLI-BglII DNA fragment encoding the 5′end of the polyprotein from Met₁ up to Leu₉₀₆, beginning at nucleotide−6 relative to Met₁. This includes the core protein (C), the E1 protein(also sometimes referred to as S), the E2 protein (also referred to asNSI), and a 5′ portion of the NS2a region. Upon expression of theconstruct, the individual C, E1 and E2 proteins are produced byproteolytic processing.

Cassette C-E1 (S/B) (a StuI-BamHI portion) contains the 5′ end from Met₁up to Ile₃₄₀, (a BamHI site in the gene). Expression of this cassetteresults in expression of C and a somewhat truncated E1 (E1′). Theportion truncated from the 3′ end is a hydrophobic region believed toserve as a translocation signal.

Cassette NS 1 (B/B) (a BamHI-BglII portion) contains a small 3′ portionof E1 (from Met₃₆₄), all of E2, and a portion of NS2a (to Leu₉₀₆). Inthis construct, the E1 fragment serves as a translocation signal.

Cassette TPA-NS1 employs a human tissue plasminogen activator (tPA)leader as a translocation signal instead of the 3′ portion of E1. Thecassette contains a truncated form of E2, from Gly₄₀₆ to Glu₆₆₁ in whichthe hydrophobic 3′ end is deleted.

Each cassette was inserted into the vector pGEM3Z (Promega) with andwithout a synthetic β-globin 5′ non-coding sequence for transcriptionand translation using T7 and rabbit reticulocyte expression in vitro.Recombinant vaccinia virus (rVV) vectors were prepared by inserting thecassettes into the plasmid pSC11 (obtained from Dr. B. Moss, NIH)followed by recombination with vaccinia virus, as described byCharkrabarty et al, Mol Cell Biol (1985) 5:3403-09.

(B) An alternate expression vector was constructed by inserting HCV(A/B)between the StuI and SpeI sites of pSC59 (obtained from Dr. B. Moss,NIH) followed by recombination with vaccinia virus, as described byCharkrabarty et al, Mol Cell Biol (1985) 5:3403-09.

(C) HeLa S3 cells were collected by centrifugation for 7 minutes at 2000rpm at room temperature in sterile 500 ml centrifuge bottles (JA-10rotor). The pellets were resuspended at a final concentration of 2×10⁷cells/ml in additional culture medium (Joklik modified MEM Spinnermedium +5% horse serum and Gentamycin) (“spinner medium”). Sonicatedcrude vv/SC59-HCV virus stock was added at a multiplicity of infectionof 8 pfu/cell, and the mixture stirred at 37° C. for 30 minutes. Theinfected cells were then transferred to a spinner flask containing 8liters spinner medium and incubated for 3 days at 37° C.

The cultured cells were then collected by centrifugation, and thepellets resuspended in buffer (10 mM Tris-HCl, pH 9.0, 15 ml). The cellswere then homogenized using a 40 ml Dounce Homogenizer (50 strokes), andthe nuclei pelleted by centrifugation (5 minutes, 1600 rpm, 4° C., JA-20rotor). The nuclear pellets were resuspended in Tris buffer (4 ml),rehomogenized, and pelleted again, pooling all supernatants.

The pooled lysate was divided into 10 ml aliquots and sonicated 3×30minutes in a cuphorn sonicator at medium power. The sonicated lysate (15ml) was layered onto 17 ml sucrose cushions (36%) in SW28 centrifugetubes, and centrifuged at 13,500 rpm for 80 minutes at 4° C. to pelletthe virus. The virus pellet was resuspended in 1 ml of Tris buffer (1 mMTris-HCl, pH 9.0) and frozen at −80° C.

Example 2 (Comparison of In Vitro and In Vivo Products)

(A) E1 and E2 were expressed both in vitro and in vivo and ³⁵S-Metlabeled using the vectors described in Example 1 above. BSC-40 and HeLacells were infected with the rVV vectors for in vivo expression. Boththe medium and the cell lysates were examined for recombinant proteins.The products were irnmunoprecipitated using human HCV immune serum,while in vitro proteins were analyzed directly. The resulting proteinswere analyzed by SDS-PAGE.

The reticulocyte expression system (pGEM3Z with HCV(S/B) or HCV(A/B))produced C, E1 and E2 proteins having molecular weights of approximately18 kD, 35 kD, and 72 kD, respectively. Lysates from BSC-40 and HeLacells transfected with rVV containing HCV(S/B), HCV(A/B) or C-E1 (S/B)exhibited the same proteins. Because the reticulocyte system does notprovide efficient golgi processing and therefore does not provide sialicacid, the fact that both in vitro and in vivo products exhibitedidentical mobilities suggests that the proteins are not sialylated invivo. Only the rVV vector containing TPA-NS1 resulted in anyextracellular secretion of E2, which exhibited an altered mobilityconsistent with sialylation.

(B) HCV(S/B) was expressed in vitro and incubated with a panel ofbiotinylated lectins: GNA, SNA, PNA, WGA, and ConA. Followingincubation, the complexes were collected on avidin-acrylic beads,washed, eluted with Laemmli sample buffer, and analyzed by SDS-PAGE. Theresults showed that E1 and E2 bound to GNA and ConA, which indicates thepresence of mannose. GNA binds to terminal mannose groups, while ConAbinds to any α-linked mannose. The lack of binding to SNA, PNA, and WGAindicates that none of the proteins contained sialic acid,galactose-N-acetylgalactosamine, or N-acetylglucosamine.

(C) Radiolabeled E1 and E2 were produced in BSC-40 cells by infectionwith rVV containing HCV(S/B) (vv/SC 11-HCV), and immunoprecipitated withhuman HCV⁺ immune serum. One half of the immunoprecipitated material wastreated overnight with neuraminidase to remove any sialic acid.Following treatment, the treated and untreated proteins were analyzed bySDS-PAGE. No significant difference in mobility was observed, indicatinglack of sialylation in vivo.

(D) Radiolabeled E1 and E2 were produced in BSC-40 cells by infectionwith rVV containing HCV(A/B) (vv/SC59-HCV), and eitherimmunoprecipitated with human HCV⁺ serum, or precipitated usingbiotinylated GNA lectin linked to acrylic beads, using vv/SC 11 free ofHCV sequences as control. The precipitates were analyzed by SDS-PAGE.The data demonstrated that E1 and E2 were the major species ofmannose-terminated proteins in vv/SC59-HCV infected cells. GNA was asefficient as human antisera in precipitating E1 and E2 from cell culturemedium. A 25 kD component was observed, but appears to be specific tovaccinia-infected cells.

Example 3 (Purification Using Lectin)

(A) HeLa S3 cells were inoculated with purified high-titer vv/SC59-HCVvirus stock at a multiplicity of infection of 5 pfu/cell, and themixture stirred at 37° C. for 30 minutes. The infected cells were thentransferred to a spinner flask containing 8 liters spinner medium andincubated for 3 days at 37° C. The cells were collected again bycentrifugation and resuspended in hypotonic buffer (20 mM HEPES, 10 mMNaCl, 1 mM MgCl₂, 120 ml) on ice. The cells were then homogenized byDounce Homogenizer (50 strokes), and the nuclei pelleted bycentrifugation (5 minutes, 1600 rpm, 4° C., JA-20 rotor). The pelletswere pooled, resuspended in 48 ml hypotonic buffer, rehomogenized,recentrifuged, pooled again, and frozen at −80° C.

The frozen supernatants were then thawed, and the microsomal membranefraction of the post-nuclear lysate isolated by centrifuging for 20minutes in a JA-20 rotor at 13,500 rpm at 4° C. The supernatant wasremoved by aspiration.

The pellets were taken up in 96 ml detergent buffer (20 mM Tris-HCl, 100mM NaCl, 1 mM EDTA, 1 mM DDT, 0.5% Triton X-100, pH 7.5) and homogenized(50 strokes). The product was clarified by centrifugation for 20 minutesat 13,500 rpm, 4° C., and the supernatants collected.

A GNA-agarose column (1 cm×3 cm, 3 mg GNA/ml beads, 6 ml bed volume,Vector Labs, Burlingame, CA) was pre-equilibrated with detergent buffer.The supernatant sample was applied to the column with recirculation at aflow rate of 1 ml/min for 16-20 hours at 4° C. The column was thenwashed with detergent buffer.

The purified E1/E2 proteins were eluted with α-D-mannoside (0.9 M indetergent buffer) at a flow rate of 0.5 ml/minute. Elution was halted atthe appearance of E1/E2 in the eluent, and the column allowed toreequilibrate for 2-3 hours. Fractions were analyzed by Western blot andsilver staining. Peak fractions were pooled and UV-irradiated toinactivate any residual vaccinia virus.

(B) GNA-agarose purified E1 and E2 asialoglycoproteins were sedimentedthrough 20-60% glycerol gradients. The gradients were fractionated andproteins were analyzed by SDS-PAGE and western blotting. Blots wereprobed with GNA for identification of E1 and E2. The results indicatethe presence of a E1:E2 heterodimer which sediments at the expected rate(i.e., a position characteristic of a 110 kD protein). Larger aggregatesof HCV envelope proteins also are apparent. E2:E2 homodimers also wereapparent. E2 appeared to be over-represented in the larger speciesrelative to E1, although discrete E1:E2 species also were detected. Thelarger aggregates sedimented significantly faster than the thyroglobulinmarker.

(C) GNA-agarose purified E1 and E2 were sedimented through 20-60%glycerol gradients containing 1 mM EDTA. Fractions were analyzed bySDS-PAGE with and without β-mercaptoethanol (βME). Little or nodifference in the apparent abundance of E1 and E2 in the presence orabsence of OME was observed, indicating the absence of disulfide linksbetween heterodimers.

(D) E1/E2 complexes (approximately 40% pure) were analyzed on a CoulterDM-4 sub-micron particle analyzer. Material in the 20-60 nm range wasdetected.

(E) E1/E2 complexes (approximately 40% pure) were analyzed by electronmicroscopy using negative staining with phosphotungstic acid. Theelectron micrograph revealed the presence of particles having aspherical appearance and a diameter of about 40 nm. E1/E2 complexes wereincubated with HCV⁺ human immune serum, then analyzed by EM withnegative staining. Antibody complexes containing large aggregates andsmaller particles were observed.

Example 4 (Chromatographic Purification)

(A) The GNA lectin-purified material prepared as described in Example 3(0.5-0.8 ml) was diluted 10× with buffer A (20 mM Tris-Cl buffer, pH8.0, 1 mM EDTA), and applied to a 1.8×1.5 cm column of Fractogel EMDDEAE-650 (EM Separations, Gibbstown, N.J., cat. no. 16883) equilibratedin buffer A. The protein fraction containing E1/E2 was eluted with thesame buffer at a flow rate of 0.2 ml/minute, and 1 ml fractionscollected. Fractions containing E1 and E2 (determined by SDS-PAGE) werepooled and stored at −80° C.

(B) The material purified in part (A) above has a purity of 60-80%, asestimated by SDS-PAGE. The identification of the putative E1 and E2bands was confirmed by N-terminal sequence analysis after using atransfer technique. For the purpose, the fractogel-DEAE purified E1/E2material was reduced by addition of Laemmli buffer (pH 6.8, 0.06 MTris-Cl, 2.3% SDS, 10% glycerol, 0.72 M β-mercaptoethanol) and boiledfor 3 minutes. The sample was then loaded onto a 10% polyacrylamide gel.After SDS-PAGE, the protein was transferred to a polyvinylidenedifluoride (PVDF) 0.2 μm membrane (Bio-Rad Laboratories, Richmond,Calif.). The respective putative E1 and E2 protein bands were excisedfrom the blot and subjected to N-terminal amino acid analysis, althoughno special care was taken to prevent amino-terminal blockage duringpreparation of the material. The first 15 cycles revealed that the E1sample had a sequenceTyr-Gln-Val-Arg-X-Ser-Thr-Gly-X-Tyr-His-Val-X-Asn-Asp, SEQ ID No: 2while the sequence of E2 wasThr-His-Val-Thr-Gly-X-X-Ala-Gly-His-X-Val-X-Gly-Phe SEQ ID No: 2. Thisamino acid sequence data is in agreement with that expected from thecorresponding DNA sequences.

The E1/E2 product purified above by fractogel-DEAE chromatography isbelieved to be aggregated as evidenced by the fact that a large amountof E1 and E2 coelutes in the void volume region of a gel permeationchromatographic Bio-Sil TSK-4000 SW column. This indicates that undernative conditions a significant amount of the E1/E2 complex has amolecular weight of at least 800 kD. E1/E2 material having a molecularweight of about 650 kD was also observed.

Example 5 (Additional Cloning and Expression)

(A) The following cassettes containing 5′ portions of the HCVpolyprotein were inserted into the vector pGEM4Z (Promega) with andwithout a synthetic yellow fever virus 5′ non-coding sequence and alsointo recombinant vaccinia virus (rVV) vectors (as described in Example1A). Cassette C5p-l contains a fragment encoding the 5′ end of thepolyprotein from Met₁ to Trp₁₀₇₉, beginning at nucleotide −275 relativeto Met₁, with EcoRI linkers on the 5′ and 3′ ends. Cassette C5p-3contains an fragment encoding the 5′ end of the polyprotein from Met₁ toTrp,₁₀₇₉, with EcoRI linkers on the 5′ and 3′ ends. Both cassettesencode C, E1 and E2 proteins and a 5′ portion of the NS2 protein.

(B) The following cassettes containing 5′ portions of the HCVpolyprotein were inserted into the vector pSC59 followed byrecombination with vaccinia virus (described in Example 1B). CassetteHCV(Poly) contains a blunt-ended StuI-BglII fragment encoding the 5′ endof the polyprotein from Met₁ to Asp₉₆₆ beginning at nucleotide −65relative to Met₁. This construct expresses C, E1 and E2 proteins, and a5′ portion of the NS2 protein.

Cassette HCV(5C/SB) contains a blunt-ended StuI-BamHI fragment encodingthe 5′ end of the polyprotein from Met₁ to Ile₃₄₀ beginning atnucleotide −65 relative to Met₁. This construct expresses the C proteinand a truncated E1 protein. Cassette HCV(6C/SS) contains a SalI(blunted)-EcoRI fragment encoding the 5′ end of the polyprotein fromMet₁ to Asp₃₈₂ wherein Ser₂ is replaced with Gly₂. This constructexpresses the C protein and a truncated E1 protein.

Cassette HCV(E12C/B) contains a blunt-ended ClaI-BglII fragment encodinga portion of the polyprotein from Met₁₃₄ to Asp₉₆₆ inserted into anEcoRI blunted SC59 vector.

Cassette HCV(E1/S) contains a blunt-ended ClaI/SalI fragment encoding aportion of the polyprotein from Met₁₃₄ to Val₃₈₁ inserted into an EcoRiblunted SC59 vector.

(C) HeLa S3 cells were collected by centrifugation for 7 minutes at 2000rpm at 4° C. in sterile 250 ml centrifuge bottles. The pellets wereresuspended at a final concentration of 5×106 cells/ml in Gey's balancedSalt Solution (GBSS). Sonicated crude vv/SC59-HCV virus stock was addedat a multiplicity of infection of 0.5 pfu/cell, and the mixture stirredat 37° C. for 1-2 hours. The infected cells were then transferred at afinal concentration of 106 cells/ml to a spinner flask containing Iliter culture medium (Joklik MEM+10% fetal bovine serum+non-essentialamino acids, vitamins, pen/strep) and incubated for 3 days at 37° C.

The cultured cells were then collected by centrifugation, and thepellets resuspended in buffer (10 mM Tris-HCl, pH 9.0, 15 ml). The cellswere then homogenized using a 40 ml Dounce Homogenizer (50 strokes), andthe nuclei pelleted by centrifugation (5 minutes, 1600 rpm, 4° C., JA-20rotor). The nuclear pellets were resuspended in Tris buffer (4 ml),rehomogenized, and pelleted again, pooling all supernatants.

The pooled lysate was divided into 5 ml aliquots, and 0.1 volume of 2.5mg/ml trypsin added and incubated at 37° C. for 30 minutes. The aliquotswere then sonicated 3×30 seconds in a cuphorn sonicator at medium power.The sonicated lysate was used as the crude stock.

Example 6 (Additional Comparison of In Vitro and In Vivo Products)

(A) E1 and E2 were expressed both in vitro and in vivo and ³⁵S-Metlabeled using the vectors described in Example 5 above and theprocedures described in Example 2 above. BSC-40 and HeLa cells wereinfected with the rVV vectors for in vivo expression. Both the mediumand the cell lysates were examined for recombinant proteins. Theproducts were immunoprecipitated using human HCV immune serum or rabbitor goat anti-HCV antiserum, while in vitro proteins were analyzeddirectly. The resulting proteins were analyzed by SDS-PAGE and EndoHdigestion.

Example 7 (Additional Lectin Purification)

(A) HeLa S3 cells were inoculated with purified high-titer vv/SC59-HCVvirus stock (HCV(Poly) or HCV(E12C/B) as described in Example 5 above)at a multiplicity of infection of 1 pfu/cell, and the mixture stirred at37° C. for 1-2 hours. The infected cells were then transferred tospinner flasks containing 1 liter culture medium (see Example 5, supra)and incubated for 2 days at 37° C. A total of 10 liters of cells werecollected by centrifugation and resuspended in hypotonic buffer (20 mMHEPES, 10 mM NaCl, 1 mM MgCl₂, 120 ml) containing protease inhibitors(PMSF and pepstatin A) on ice. The cells were then homogenized in a 40ml homogenizer in two batches, pelleted by centrifugation (20 minutes,12,000 rpm), and re-suspended and re-homogenized. Each pellet wasresuspended in approximately 10 ml 25 mM NaPO₄ (pH 6.8) in ahomogenizer, and an equal volume of 4% Triton X-100 in 100 mM NaPO₄ (pH6.8) added. The pelleted cells were homogenized with 20 strokes, spun at12,000 rpm for 15 minutes (4 tubes/pellet), and the supernatant saved.The resuspension, Triton addition and centrifugation steps wererepeated, and the saved supernatants combined, and frozen at −80° C.

The frozen supernatants were thawed, spun at 12,000 rpm for 15 minutes,combined and held at 4° C. A GNA-agarose column (1 cm×3 cm, 3 mg GNA/mlbeads, 6 ml bed volume, Vector Labs, Burlingame, Calif.) waspre-equilibrated with detergent buffer (2% Triton X-100 in 50 mM NaPO₄,pH 6.8). The supernatant sample was applied to the column withrecirculation at a flow rate of 1 ml/min for 16-20 hours at 4° C. Thecolumn was then washed with detergent buffer, followed by 30 ml each of:Buffer A (1M NaCl, 20 mM NaPO₄ (pH 6.0), 0.1% Triton X-100); Buffer B(20 mM NaPO₄ (pH 6.0), 0.1% Triton X-100); Buffer D (0.2Mmethyl-α-D-mannopyranoside (mmp), 20 mM NaPO₄ (pH 6.0), 0.1% TritonX-100); Buffer E (1M mmp, 20 mM NaPO₄ (pH 6.0), 0.1% Triton X-100);Buffer F (1M mmp, 1M NaCl, 20 mM NaPO₄ (pH 6.0), 0.1% Triton X-100).Purified E1/E2 proteins come off as eluted material in Buffers D and E,which were collected separately and analyzed by SDS-PAGE.

Example 8 (Additional Chromatographic Purification)

(A) The GNA lectin-purified material prepared as described in Example 7was applied to a column of S-Sepharose Fast Flow (Pharmacia)equilibrated in buffer B (see Example 7). The column was washed withBuffer B, then eluted with Buffer 1 (0.5M NaCl, 20 mM NaPO₄ (pH 6.0),0.1% Triton X-100) and Buffer 2 (1M NaCl, 20 mM NaPO₄ (pH 6.0), 0.1%Triton X-100). Fractions containing E1 and E2 (determined by SDS-PAGE)were pooled and stored at −80° C.

3 15 amino acids amino acid single linear protein not provided 1 Tyr GlnVal Arg Xaa Ser Thr Gly Xaa Tyr His Val Xaa Asn Asp 1 5 10 15 15 aminoacids amino acid single linear protein not provided 2 Thr His Val ThrGly Xaa Xaa Ala Gly His Xaa Val Xaa Gly Phe 1 5 10 15 2955 amino acidsamino acid single linear protein not provided Modified-site /note=“There is a heterogeneity at this location; Xaa = Arg or Lys”Modified-site 11 /note= “There is a heterogeneity at this location; Xaa= Asn or Thr” Modified-site 176 /note= “There is a heterogeneity at thislocation; Xaa = Ile or Thr” Modified-site 334 /note= “There is aheterogeneity at this location; Xaa = Met or Val” Modified-site 603/note= “There is a heterogeneity at this location; Xaa = Ile or Leu”Modified-site 848 /note= “There is a heterogeneity at this location; Xaa= Asn or Tyr” Modified-site 1114 /note= “There is a heterogeneity atthis location; Xaa = Pro or Ser” Modified-site 1117 /note= “There is aheterogeneity at this location; Xaa = Ser or Thr” Modified-site 1276/note= “There is a heterogeneity at this location; Xaa = Leu or Pro”Modified-site 1454 /note= “There is a heterogeneity at this location;Xaa = Cys or Tyr” Modified-site 1471 /note= “There is a heterogeneity atthis location; Xaa = Ser or Thr” Modified-site 1877 /note= “There is aheterogeneity at this location; Xaa = Glu or Gly” Modified-site 1948/note= “There is a heterogeneity at this location; Xaa = His or Leu”Modified-site 1949 /note= “There is a heterogeneity at this location;Xaa = Cys or Ser” Modified-site 2021 /note= “There is a heterogeneity atthis location; Xaa = Gly or Val” Modified-site 2349 /note= “There is aheterogeneity at this location; Xaa = Ser or Thr” Modified-site 2385/note= “There is a heterogeneity at this location; Xaa =Phe or Tyr”Modified-site 2386 /note= “There is a heterogeneity at this location;Xaa = Ala or Ser” Modified-site 2502 /note= “There is a heterogeneity atthis location; Xaa = Phe or Leu” Modified-site 2690 /note= “There is aheterogeneity at this location; Xaa = Gly or Arg” Modified-site 2921/note= “There is a heterogeneity at this location; Xaa = Arg or Gly” 3Met Ser Thr Asn Pro Lys Pro Gln Xaa Lys Xaa Lys Arg Asn Thr Asn 1 5 1015 Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly 20 2530 Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala 35 4045 Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro 50 5560 Ile Pro Lys Ala Arg Arg Pro Glu Gly Arg Thr Trp Ala Gln Pro Gly 65 7075 80 Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Cys Gly Trp Ala Gly Trp 8590 95 Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro100 105 110 Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu ThrCys 115 120 125 Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu Val Gly AlaPro Leu 130 135 140 Gly Ser Ala Ala Arg Ala Leu Ala His Gly Val Arg ValLeu Glu Asp 145 150 155 160 Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro GlyCys Ser Phe Ser Xaa 165 170 175 Phe Leu Leu Ala Leu Leu Ser Cys Leu ThrVal Pro Ala Ser Ala Tyr 180 185 190 Gln Val Arg Asn Ser Thr Gly Leu TyrHis Val Thr Asn Asp Cys Pro 195 200 205 Asn Ser Ser Ile Val Tyr Glu AlaAla Asp Ala Ile Leu His Thr Pro 210 215 220 Gly Cys Val Pro Cys Val ArgGlu Gly Asn Ala Ser Arg Cys Trp Val 225 230 235 240 Ala Met Thr Pro ThrVal Ala Thr Arg Asp Gly Lys Leu Pro Ala Thr 245 250 255 Gln Leu Arg ArgHis Ile Asp Leu Leu Val Gly Ser Ala Thr Leu Cys 260 265 270 Ser Ala LeuTyr Val Gly Asp Leu Cys Gly Ser Val Phe Leu Val Gly 275 280 285 Gln LeuPhe Thr Phe Ser Pro Arg Arg His Trp Thr Thr Gln Gly Cys 290 295 300 AsnCys Ser Ile Tyr Pro Gly His Ile Thr Gly His Arg Met Ala Trp 305 310 315320 Asp Met Met Met Asn Trp Ser Pro Thr Thr Ala Leu Val Xaa Ala Gln 325330 335 Leu Leu Arg Ile Pro Gln Ala Ile Leu Asp Met Ile Ala Gly Ala His340 345 350 Trp Gly Val Leu Ala Gly Ile Ala Tyr Phe Ser Met Val Gly AsnTrp 355 360 365 Ala Lys Val Leu Val Val Leu Leu Leu Phe Ala Gly Val AspAla Glu 370 375 380 Thr His Val Thr Gly Gly Ser Ala Gly His Thr Val SerGly Phe Val 385 390 395 400 Ser Leu Leu Ala Pro Gly Ala Lys Gln Asn ValGln Leu Ile Asn Thr 405 410 415 Asn Gly Ser Trp His Leu Asn Ser Thr AlaLeu Asn Cys Asn Asp Ser 420 425 430 Leu Asn Thr Gly Trp Leu Ala Gly LeuPhe Tyr His His Lys Phe Asn 435 440 445 Ser Ser Gly Cys Pro Glu Arg LeuAla Ser Cys Arg Pro Leu Thr Asp 450 455 460 Phe Asp Gln Gly Trp Gly ProIle Ser Tyr Ala Asn Gly Ser Gly Pro 465 470 475 480 Asp Gln Arg Pro TyrCys Trp His Tyr Pro Pro Lys Pro Cys Gly Ile 485 490 495 Val Pro Ala LysSer Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser 500 505 510 Pro Val ValVal Gly Thr Thr Asp Arg Ser Gly Ala Pro Thr Tyr Ser 515 520 525 Trp GlyGlu Asn Asp Thr Asp Val Phe Val Leu Asn Asn Thr Arg Pro 530 535 540 ProLeu Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Ser Thr Gly Phe 545 550 555560 Thr Lys Val Cys Gly Ala Pro Pro Cys Val Ile Gly Gly Ala Gly Asn 565570 575 Asn Thr Leu His Cys Pro Thr Asp Cys Phe Arg Lys His Pro Asp Ala580 585 590 Thr Tyr Ser Arg Cys Gly Ser Gly Pro Trp Xaa Thr Pro Arg CysLeu 595 600 605 Val Asp Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr IleAsn Tyr 610 615 620 Thr Ile Phe Lys Ile Arg Met Tyr Val Gly Gly Val GluHis Arg Leu 625 630 635 640 Glu Ala Ala Cys Asn Trp Thr Arg Gly Glu ArgCys Asp Leu Glu Asp 645 650 655 Arg Asp Arg Ser Glu Leu Ser Pro Leu LeuLeu Thr Thr Thr Gln Trp 660 665 670 Gln Val Leu Pro Cys Ser Phe Thr ThrLeu Pro Ala Leu Ser Thr Gly 675 680 685 Leu Ile His Leu His Gln Asn IleVal Asp Val Gln Tyr Leu Tyr Gly 690 695 700 Val Gly Ser Ser Ile Ala SerTrp Ala Ile Lys Trp Glu Tyr Val Val 705 710 715 720 Leu Leu Phe Leu LeuLeu Ala Asp Ala Arg Val Cys Ser Cys Leu Trp 725 730 735 Met Met Leu LeuIle Ser Gln Ala Glu Ala Ala Leu Glu Asn Leu Val 740 745 750 Ile Leu AsnAla Ala Ser Leu Ala Gly Thr His Gly Leu Val Ser Phe 755 760 765 Leu ValPhe Phe Cys Phe Ala Trp Tyr Leu Lys Gly Lys Trp Val Pro 770 775 780 GlyAla Val Tyr Thr Phe Tyr Gly Met Trp Pro Leu Leu Leu Leu Leu 785 790 795800 Leu Ala Leu Pro Gln Arg Ala Tyr Ala Leu Asp Thr Glu Val Ala Ala 805810 815 Ser Cys Gly Gly Val Val Leu Val Gly Leu Met Ala Leu Thr Leu Ser820 825 830 Pro Tyr Tyr Lys Arg Tyr Ile Ser Trp Cys Leu Trp Trp Leu GlnXaa 835 840 845 Phe Leu Thr Arg Val Glu Ala Gln Leu His Val Trp Ile ProPro Leu 850 855 860 Asn Val Arg Gly Gly Arg Asp Ala Val Ile Leu Leu MetCys Ala Val 865 870 875 880 His Pro Thr Leu Val Phe Asp Ile Thr Lys LeuLeu Leu Ala Val Phe 885 890 895 Gly Pro Leu Trp Ile Leu Gln Ala Ser LeuLeu Lys Val Pro Tyr Phe 900 905 910 Val Arg Val Gln Gly Leu Leu Arg PheCys Ala Leu Ala Arg Lys Met 915 920 925 Ile Gly Gly His Tyr Val Gln MetVal Ile Ile Lys Leu Gly Ala Leu 930 935 940 Thr Gly Thr Tyr Val Tyr AsnHis Leu Thr Pro Leu Arg Asp Trp Ala 945 950 955 960 His Asn Gly Leu ArgAsp Leu Ala Val Ala Val Glu Pro Val Val Phe 965 970 975 Ser Gln Met GluThr Lys Leu Ile Thr Trp Gly Ala Asp Thr Ala Ala 980 985 990 Cys Gly AspIle Ile Asn Gly Leu Pro Val Ser Ala Arg Arg Gly Arg 995 1000 1005 GluIle Leu Leu Gly Pro Ala Asp Gly Met Val Ser Lys Gly Trp Arg 1010 10151020 Leu Leu Ala Pro Ile Thr Ala Tyr Ala Gln Gln Thr Arg Gly Leu Leu1025 1030 1035 1040 Gly Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp Lys AsnGln Val Glu 1045 1050 1055 Gly Glu Val Gln Ile Val Ser Thr Ala Ala GlnThr Phe Leu Ala Thr 1060 1065 1070 Cys Ile Asn Gly Val Cys Trp Thr ValTyr His Gly Ala Gly Thr Arg 1075 1080 1085 Thr Ile Ala Ser Pro Lys GlyPro Val Ile Gln Met Tyr Thr Asn Val 1090 1095 1100 Asp Gln Asp Leu ValGly Trp Pro Ala Xaa Gln Gly Xaa Arg Ser Leu 1105 1110 1115 1120 Thr ProCys Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr Arg His 1125 1130 1135Ala Asp Val Ile Pro Val Arg Arg Arg Gly Asp Ser Arg Gly Ser Leu 11401145 1150 Leu Ser Pro Arg Pro Ile Ser Tyr Leu Lys Gly Ser Ser Gly GlyPro 1155 1160 1165 Leu Leu Cys Pro Ala Gly His Ala Val Gly Ile Phe ArgAla Ala Val 1170 1175 1180 Cys Thr Arg Gly Val Ala Lys Ala Val Asp PheIle Pro Val Glu Asn 1185 1190 1195 1200 Leu Glu Thr Thr Met Arg Ser ProVal Phe Thr Asp Asn Ser Ser Pro 1205 1210 1215 Pro Val Val Pro Gln SerPhe Gln Val Ala His Leu His Ala Pro Thr 1220 1225 1230 Gly Ser Gly LysSer Thr Lys Val Pro Ala Ala Tyr Ala Ala Gln Gly 1235 1240 1245 Tyr LysVal Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu Gly Phe 1250 1255 1260Gly Ala Tyr Met Ser Lys Ala His Gly Ile Asp Xaa Asn Ile Arg Thr 12651270 1275 1280 Gly Val Arg Thr Ile Thr Thr Gly Ser Pro Ile Thr Tyr SerThr Tyr 1285 1290 1295 Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly GlyAla Tyr Asp Ile 1300 1305 1310 Ile Ile Cys Asp Glu Cys His Ser Thr AspAla Thr Ser Ile Leu Gly 1315 1320 1325 Ile Gly Thr Val Leu Asp Gln AlaGlu Thr Ala Gly Ala Arg Leu Val 1330 1335 1340 Val Leu Ala Thr Ala ThrPro Pro Gly Ser Val Thr Val Pro His Pro 1345 1350 1355 1360 Asn Ile GluGlu Val Ala Leu Ser Thr Thr Gly Glu Ile Pro Phe Tyr 1365 1370 1375 GlyLys Ala Ile Pro Leu Glu Val Ile Lys Gly Gly Arg His Leu Ile 1380 13851390 Phe Cys His Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys Leu Val1395 1400 1405 Ala Leu Gly Ile Asn Ala Val Ala Tyr Tyr Arg Gly Leu AspVal Ser 1410 1415 1420 Val Ile Pro Thr Ser Gly Asp Val Val Val Val AlaThr Asp Ala Leu 1425 1430 1435 1440 Met Thr Gly Tyr Thr Gly Asp Phe AspSer Val Ile Asp Xaa Asn Thr 1445 1450 1455 Cys Val Thr Gln Thr Val AspPhe Ser Leu Asp Pro Thr Phe Xaa Ile 1460 1465 1470 Glu Thr Ile Thr LeuPro Gln Asp Ala Val Ser Arg Thr Gln Arg Arg 1475 1480 1485 Gly Arg ThrGly Arg Gly Lys Pro Gly Ile Asn Arg Phe Val Ala Pro 1490 1495 1500 GlyGlu Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys Glu Cys 1505 15101515 1520 Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala Glu ThrThr 1525 1530 1535 Val Arg Leu Arg Ala Tyr Met Asn Thr Pro Gly Leu ProVal Cys Gln 1540 1545 1550 Asp His Leu Glu Phe Trp Glu Gly Val Phe ThrGly Leu Thr His Ile 1555 1560 1565 Asp Ala His Phe Leu Ser Gln Thr LysGln Ser Gly Glu Asn Leu Pro 1570 1575 1580 Tyr Leu Val Ala Tyr Gln AlaThr Val Cys Ala Arg Ala Gln Ala Pro 1585 1590 1595 1600 Pro Pro Ser TrpAsp Gln Met Trp Lys Cys Leu Ile Arg Leu Lys Pro 1605 1610 1615 Thr LeuHis Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val Gln 1620 1625 1630Asn Glu Ile Thr Leu Thr His Pro Val Thr Lys Tyr Ile Met Thr Cys 16351640 1645 Met Ser Ala Asp Leu Glu Val Val Thr Ser Thr Trp Val Leu ValGly 1650 1655 1660 Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu Ser ThrGly Cys Val 1665 1670 1675 1680 Val Ile Val Gly Arg Val Val Leu Ser GlyLys Pro Ala Ile Ile Pro 1685 1690 1695 Asp Arg Glu Val Leu Tyr Arg GluPhe Asp Glu Met Glu Glu Cys Ser 1700 1705 1710 Gln His Leu Pro Tyr IleGlu Gln Gly Met Met Leu Ala Glu Gln Phe 1715 1720 1725 Lys Gln Lys AlaLeu Gly Leu Leu Gln Thr Ala Ser Arg Gln Ala Glu 1730 1735 1740 Val IleAla Pro Ala Val Gln Thr Asn Trp Gln Lys Leu Glu Thr Phe 1745 1750 17551760 Trp Ala Lys His Met Trp Asn Phe Ile Ser Gly Ile Gln Tyr Leu Ala1765 1770 1775 Gly Leu Ser Thr Leu Pro Gly Asn Pro Ala Ile Ala Ser LeuMet Ala 1780 1785 1790 Phe Thr Ala Ala Val Thr Ser Pro Leu Thr Thr SerGln Thr Leu Leu 1795 1800 1805 Phe Asn Ile Leu Gly Gly Trp Val Ala AlaGln Leu Ala Ala Pro Gly 1810 1815 1820 Ala Ala Thr Ala Phe Val Gly AlaGly Leu Ala Gly Ala Ala Ile Gly 1825 1830 1835 1840 Ser Val Gly Leu GlyLys Val Leu Ile Asp Ile Leu Ala Gly Tyr Gly 1845 1850 1855 Ala Gly ValAla Gly Ala Leu Val Ala Phe Lys Ile Met Ser Gly Glu 1860 1865 1870 ValPro Ser Thr Xaa Asp Leu Val Asn Leu Leu Pro Ala Ile Leu Ser 1875 18801885 Pro Gly Ala Leu Val Val Gly Val Val Cys Ala Ala Ile Leu Arg Arg1890 1895 1900 His Val Gly Pro Gly Glu Gly Ala Val Gln Trp Met Asn ArgLeu Ile 1905 1910 1915 1920 Ala Phe Ala Ser Arg Gly Asn His Val Ser ProThr His Tyr Val Pro 1925 1930 1935 Glu Ser Asp Ala Ala Ala Arg Val ThrAla Ile Xaa Xaa Ser Leu Thr 1940 1945 1950 Val Thr Gln Leu Leu Arg ArgLeu His Gln Trp Ile Ser Ser Glu Cys 1955 1960 1965 Thr Thr Pro Cys SerGly Ser Trp Leu Arg Asp Ile Trp Asp Trp Ile 1970 1975 1980 Cys Glu ValLeu Ser Asp Phe Lys Thr Trp Leu Lys Ala Lys Leu Met 1985 1990 1995 2000Pro Gln Leu Pro Gly Ile Pro Phe Val Ser Cys Gln Arg Gly Tyr Lys 20052010 2015 Gly Val Trp Arg Xaa Asp Gly Ile Met His Thr Arg Cys His CysGly 2020 2025 2030 Ala Glu Ile Thr Gly His Val Lys Asn Gly Thr Met ArgIle Val Gly 2035 2040 2045 Pro Arg Thr Cys Arg Asn Met Trp Ser Gly ThrPhe Pro Ile Asn Ala 2050 2055 2060 Tyr Thr Thr Gly Pro Cys Thr Pro LeuPro Ala Pro Asn Tyr Thr Phe 2065 2070 2075 2080 Ala Leu Trp Arg Val SerAla Glu Glu Tyr Val Glu Ile Arg Gln Val 2085 2090 2095 Gly Asp Phe HisTyr Val Thr Gly Met Thr Thr Asp Asn Leu Lys Cys 2100 2105 2110 Pro CysGln Val Pro Ser Pro Glu Phe Phe Thr Glu Leu Asp Gly Val 2115 2120 2125Arg Leu His Arg Phe Ala Pro Pro Cys Lys Pro Leu Leu Arg Glu Glu 21302135 2140 Val Ser Phe Arg Val Gly Leu His Glu Tyr Pro Val Gly Ser GlnLeu 2145 2150 2155 2160 Pro Cys Glu Pro Glu Pro Asp Val Ala Val Leu ThrSer Met Leu Thr 2165 2170 2175 Asp Pro Ser His Ile Thr Ala Glu Ala AlaGly Arg Arg Leu Ala Arg 2180 2185 2190 Gly Ser Pro Pro Ser Val Ala SerSer Ser Ala Ser Gln Leu Ser Ala 2195 2200 2205 Pro Ser Leu Lys Ala ThrCys Thr Ala Asn His Asp Ser Pro Asp Ala 2210 2215 2220 Glu Leu Ile GluAla Asn Leu Leu Trp Arg Gln Glu Met Gly Gly Asn 2225 2230 2235 2240 IleThr Arg Val Glu Ser Glu Asn Lys Val Val Ile Leu Asp Ser Phe 2245 22502255 Asp Pro Leu Val Ala Glu Glu Asp Glu Arg Glu Ile Ser Val Pro Ala2260 2265 2270 Glu Ile Leu Arg Lys Ser Arg Arg Phe Ala Gln Ala Leu ProVal Trp 2275 2280 2285 Ala Arg Pro Asp Tyr Asn Pro Pro Leu Val Glu ThrTrp Lys Lys Pro 2290 2295 2300 Asp Tyr Glu Pro Pro Val Val His Gly CysPro Leu Pro Pro Pro Lys 2305 2310 2315 2320 Ser Pro Pro Val Pro Pro ProArg Lys Lys Arg Thr Val Val Leu Thr 2325 2330 2335 Glu Ser Thr Leu SerThr Ala Leu Ala Glu Leu Ala Xaa Arg Ser Phe 2340 2345 2350 Gly Ser SerSer Thr Ser Gly Ile Thr Gly Asp Asn Thr Thr Thr Ser 2355 2360 2365 SerGlu Pro Ala Pro Ser Gly Cys Pro Pro Asp Ser Asp Ala Glu Ser 2370 23752380 Xaa Xaa Ser Met Pro Pro Leu Glu Gly Glu Pro Gly Asp Pro Asp Leu2385 2390 2395 2400 Ser Asp Gly Ser Trp Ser Thr Val Ser Ser Glu Ala AsnAla Glu Asp 2405 2410 2415 Val Val Cys Cys Ser Met Ser Tyr Ser Trp ThrGly Ala Leu Val Thr 2420 2425 2430 Pro Cys Ala Ala Glu Glu Gln Lys LeuPro Ile Asn Ala Leu Ser Asn 2435 2440 2445 Ser Leu Leu Arg His His AsnLeu Val Tyr Ser Thr Thr Ser Arg Ser 2450 2455 2460 Ala Cys Gln Arg GlnLys Lys Val Thr Phe Asp Arg Leu Gln Val Leu 2465 2470 2475 2480 Asp SerHis Tyr Gln Asp Val Leu Lys Glu Val Lys Ala Ala Ala Ser 2485 2490 2495Lys Val Lys Ala Asn Xaa Leu Ser Val Glu Glu Ala Cys Ser Leu Thr 25002505 2510 Pro Pro His Ser Ala Lys Ser Lys Phe Gly Tyr Gly Ala Lys AspVal 2515 2520 2525 Arg Cys His Ala Arg Lys Ala Val Thr His Ile Asn SerVal Trp Lys 2530 2535 2540 Asp Leu Leu Glu Asp Asn Val Thr Pro Ile AspThr Thr Ile Met Ala 2545 2550 2555 2560 Lys Asn Glu Val Phe Cys Val GlnPro Glu Lys Gly Gly Arg Lys Pro 2565 2570 2575 Ala Arg Leu Ile Val PhePro Asp Leu Gly Val Arg Val Cys Glu Lys 2580 2585 2590 Met Ala Leu TyrAsp Val Val Thr Lys Leu Pro Leu Ala Val Met Gly 2595 2600 2605 Ser SerTyr Gly Phe Gln Tyr Ser Pro Gly Gln Arg Val Glu Phe Leu 2610 2615 2620Val Gln Ala Trp Lys Ser Lys Lys Thr Pro Met Gly Phe Ser Tyr Asp 26252630 2635 2640 Thr Arg Cys Phe Asp Ser Thr Val Thr Glu Ser Asp Ile ArgThr Glu 2645 2650 2655 Glu Ala Ile Tyr Gln Cys Cys Asp Leu Asp Pro GlnAla Arg Val Ala 2660 2665 2670 Ile Lys Ser Leu Thr Glu Arg Leu Tyr ValGly Gly Pro Leu Thr Asn 2675 2680 2685 Ser Xaa Gly Glu Asn Cys Gly TyrArg Arg Cys Arg Ala Ser Gly Val 2690 2695 2700 Leu Thr Thr Ser Cys GlyAsn Thr Leu Thr Cys Tyr Ile Lys Ala Arg 2705 2710 2715 2720 Ala Ala CysArg Ala Ala Gly Leu Gln Asp Cys Thr Met Leu Val Cys 2725 2730 2735 GlyAsp Asp Leu Val Val Ile Cys Glu Ser Ala Gly Val Gln Glu Asp 2740 27452750 Ala Ala Ser Leu Arg Ala Phe Thr Glu Ala Met Thr Arg Tyr Ser Ala2755 2760 2765 Pro Pro Gly Asp Pro Pro Gln Pro Glu Tyr Asp Leu Glu LeuIle Thr 2770 2775 2780 Ser Cys Ser Ser Asn Val Ser Val Ala His Asp GlyAla Gly Lys Arg 2785 2790 2795 2800 Val Tyr Tyr Leu Thr Arg Asp Pro ThrThr Pro Leu Ala Arg Ala Ala 2805 2810 2815 Trp Glu Thr Ala Arg His ThrPro Val Asn Ser Trp Leu Gly Asn Ile 2820 2825 2830 Ile Met Phe Ala ProThr Leu Trp Ala Arg Met Ile Leu Met Thr His 2835 2840 2845 Phe Phe SerVal Leu Ile Ala Arg Asp Gln Leu Glu Gln Ala Leu Asp 2850 2855 2860 CysGlu Ile Tyr Gly Ala Cys Tyr Ser Ile Glu Pro Leu Asp Leu Pro 2865 28702875 2880 Pro Ile Ile Gln Arg Leu His Gly Leu Ser Ala Phe Ser Leu HisSer 2885 2890 2895 Tyr Ser Pro Gly Glu Ile Asn Arg Val Ala Ala Cys LeuArg Lys Leu 2900 2905 2910 Gly Val Pro Pro Leu Arg Ala Trp Xaa His ArgAla Arg Ser Val Arg 2915 2920 2925 Ala Arg Leu Leu Ala Arg Gly Gly ArgAla Ala Ile Cys Gly Lys Tyr 2930 2935 2940 Leu Phe Asn Trp Ala Val ArgThr Lys Leu Lys 2945 2950 2955

What is claimed:
 1. A hepatitis C virus (HCV) glycoprotein compositioncomprising a pharmaceutically acceptable excipient and a purified HCVE1/E2 glycoprotein aggregate, wherein said glycoproteins of saidaggregate have mannose-terminated glycosylation, wherein less than about10% of the total N-linked carbohydrate on said glycoproteins is sialicacid, and further wherein said aggregate comprises a glycoproteinexpressed from the E1 region of HCV and a glycoprotein expressed fromthe E2 region of HCV, said aggregate produced by the method comprisingthe steps of: contacting a composition containing HCV glycoproteins witha mannose-binding protein specific for mannose-terminated glycoproteins;and isolating the portion of the composition which binds to saidmannose-binding protein.
 2. The composition of claim 1 wherein saidmannose-binding protein is a lectin.
 3. The composition of claim 2wherein said lectin is Galanthus nivalus agglutinin.
 4. A compositioncomprising a pharmaceutically acceptable excipient and a purifiedhepatitis C virus (HCV) glycoprotein having mannose-terminatedglycosylation, wherein less than about 10% of the total N-linkedcarbohydrate on said HCV glycoprotein is sialie acid, wherein said HCVglycoprotein is selected from the group consisting of a glycoproteinexpressed from the E1 region of HCV and a glycoprotein expressed fromthe E2 region of HCV, produced by the method comprising the steps of:growing a mammalian host cell transformed with a structural geneencoding said HCV glycoprotein in a suitable culture medium, whereinsaid structural gene is linked to a sequence encoding a secretion leaderthat directs the glycoprotein to the endoplasmic reticulum; causingexpression of said structural gene and secretion leader sequence underconditions inhibiting sialylation, wherein said conditions inhibitingsialylation comprise inhibiting transport of glycoproteins from theendoplasmic reticulum to the golgi; and isolating said HCV glycoproteinfrom said cell culture by contacting said HCV glycoprotein with amannose-binding protein specific for mannose-terminated glycoproteins,and isolating the protein which binds to said mannose-binding protein.5. The composition of claim 4, wherein said qlycoprotein is expressedfrom the E1 region of HCV.
 6. The composition of claim 4, wherein saidglycoprotein is expressed from the E2 region of HCV.
 7. The compositionof claim 4, wherein said mannose-binding protein is a lectin.
 8. Thecomposition of claim 5, wherein said mannose-binding protein is alectin.
 9. The composition of claim 6, wherein said mannose-bindingprotein is a lectin.
 10. A method of inducing an immune response in ananimal, which method comprises: providing an effective amount of acomposition according to claim 4; and administering said composition tosaid animal.
 11. The method of claim 10, wherein said HCV glycoproteinis expressed from the E1 region of HCV.
 12. The method of claim 10,wherein said HCV glycoprotein is expressed from the E2 region of HCV.13. The method of claim 10, wherein said HCV glycoprotein is an E1/E2aggregate.
 14. The method of claim 10, wherein said animal is a primate.15. An isolated hepatitis C virus (HCV) glycoprotein havingmannose-terminated glycosylation, wherein less than about 10% of thetotal N-linked carbohydrate on said HCV glycoprotein is sialic acid,wherein said HCV glycoprotein is selected from the group consisting of aglycoprotein expressed from the E1 region of HCV, a glycoproteinexpressed fiom the E2 region of HCV, and an E1/E2 aggregateglycoprotein, wherein said glycoprotein is produced by the methodcomprising the steps of: growing a mammalian host cell transformed witha structural gene encoding said glycoprotein in a suitable culturemedium, wherein said structural gene is linked to a sequence encoding asecretion leader that directs the glycoprotein to the endoplasmicreticulum; causing expression of said structural gene and secretionleader sequence under conditions inhibiting sialylation, wherein saidconditions inhibiting sialylation comprise inhibiting transport ofglycoproteins from the endoplasmic reticulum to the golgi; and isolatingsaid HCV glycoprotein from said cell culture by contacting said HCVglycoprotein with a mannose-binding protein specific formannose-terminated glycoproteins.
 16. The HCV glycoprotein of claim 15wherein said conditions inhibiting sialylation comprise expression ofthe glycoprotein at a rate sufficient to inhibit transport of theglycoprotein from the endoplasmic reticulum to the golgi.
 17. The HCVglycoprotein of claim 15 wherein said conditions inhibiting sialylationfurther comprise a sufficient amount of a calcium modulator to causerelease of proteins within the host cell's endoplasmic reticulum. 18.The HCV glycoprotein of any of claims 15-17, wherein said glycoproteinis expressed from the E1 region of HCV.
 19. The HCV glycoprotein of anyof claims 15-17, wherein said glycoprotein is expressed from the E2region of HCV.
 20. The HCV glycoprotein of any of claims 15-17, whereinsaid glycoprotein is an E1/E2 aggregate.
 21. The HCV glycoprotein of anyof claims 15-17, wherein said glycoprotein is an E1/E1 aggregate. 22.The HCV glycoprotein of any of claims 15-17, wherein said glycoproteinis an E2/E2 aggregate.
 23. An isolated hepatitis C virus (HCV) E1/E2glycoprotein aggregate having mannose-terminated glycosylation, whereinless than about 10% of the total N-linked carbohydrate on said HCVglycoprotein is sialic acid, and further wherein said HCV glycoproteinaggregate comprises a glycoprotein expressed from the E1 region of HCVand a glycoprotein expressed from the E2 region of HCV, wherein saidglycoprotein aggregate is produced by the method comprising the stepsof: growing a host cell transformed with a structural gene encoding saidglycoprotein in a suitable culture medium; causing expression of saidstructural gene under conditions inhibiting sialylation; and isolatingsaid HCV glycoprotein aggregate from said cell culture by contactingsaid HCV glycoprotein with a mannose-binding protein specific formannose-terminated glycoproteins.
 24. A method of inducing an immuneresponse in an animal, which methods comprises: providing an effectiveamount of a composition according to claim 1; and administering saidcomposition to said animal.
 25. The method of claim 24, wherein saidanimal is a primate.